Enhanced cell/bead encapsulation methods and apparatuses

ABSTRACT

A microfluidic droplet system that employs flow-focusing methods to generate droplets of desired sizes for encapsulating particles, cells or beads. The microfluidic droplet system can comprise an air cavity that can be vibrated to reduce trapping of cells or beads in flow-focusing regions of the microfluidic droplet systems thereby increasing the encapsulation efficiency of the cells/beads.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

The inventions were made with government support under Grant No. 1362165awarded by the National Science Foundation. The government may havecertain rights in the inventions.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

Any and all applications for which a foreign or domestic priority claimis identified in the Application Data Sheet as filed with the presentapplication are hereby incorporated by reference under 37 C.F.R. §1.57.

BACKGROUND

Field of the Invention

This disclosure relates generally to microfluidic devices.

Description of the Related Art

Microfluidic devices and systems are configured to process (e.g., move,mix, separate) small volumes of fluid. Microfluidic devices and systemsare used for various applications including printing, bio-chemicalassays, drug discovery, etc. A class of microfluidic devices and systemsincludes microfluidic droplet generating and manipulating devices.Droplet-based microfluidic devices and systems are compatible with manychemical and biological reagents. Droplet-based microfluidic devices canbe configured to manipulate discrete droplets. Droplet-basedmicrofluidic devices can be configured to perform a variety ofoperations, such as, for example, transportation of droplets, storage ofdroplets, mixing of droplets, analysis of droplets, etc. Droplet-basedmicrofluidic devices can be configured to perform a variety ofoperations repeatably using a set of programmable instructions.Accordingly, droplet-based microfluidic devices can be also be referredto as digital microfluidic devices. Droplet-based microfluidic devicescan be used in a variety of applications including but not limited to asmicroreactors to achieve controlled and rapid mixing of fluids and/or tosynthesize particles and encapsulate many biological entities forbiomedicine and biotechnology applications.

SUMMARY

Example embodiments described herein have several features, no singleone of which is indispensable or solely responsible for their desirableattributes. Without limiting the scope of the claims, some of theadvantageous features will now be summarized.

Various embodiments discussed herein include droplet-based microfluidicdevices that rely on trapped air bubbles in liquid to disruptparticle-trapping vortices and facilitate cell/bead encapsulation. Asmicrofluidic droplet production rate is increased, and size isdecreased, incoming cells/beads tend to become trapped in microvorticesin the flow-focusing region, instead of being encapsulated in thedroplets. To disrupt the microvortices in the flow-focusing region, anair cavity is integrated in the droplet generation region of amicrofluidic device. The air cavity can be integrated upstream from anorifice of the flow-focusing region that generates the liquid droplets.During droplet generation, the air trapped in the structure can bevibrated. The vibrations can produce vortices that flow counter to themicrovortices produced in the flow-focusing region of the microfluidicdevice. Accordingly, devices including the concepts described herein canreduce cell/bead trapping upstream from the orifice of the flow-focusingregion and improve encapsulation efficiency.

Various embodiments discussed herein include a microfluidic devicecomprising an acoustically resonant structure that can improve cell/beadencapsulation efficiency. It may be possible to switch between acell/bead trapping mode in which cells/beads are trapped upstream froman orifice of the flow-focusing region, and an encapsulating mode inwhich the cell/bead are encapsulated in droplets by controllingpiezoelectric transducer activation. The embodiments described hereincan increase the percentage of cells/beads encapsulated in dropletswhich can be useful in the field of single cell and single analyteassays. Single cell assay platforms with vibrating air cavitiescontrolled by piezoelectric transducer can be easier to use and cheaperto manufacture than single cell assay platforms that rely on magneticbead or cytometry-based switching approaches. Single cell assayplatforms with vibrating air cavities controlled by piezoelectrictransducer can also improve the percentage of cells/beads that areencapsulated in droplets over existing methods (e.g., magnetic bead orcytometry-based approaches) that may affect outcome of single cellassays. Single cell assay platforms with vibrating air cavitiescontrolled by piezoelectric transducer can be used to carry outgenotyping or gene sequencing on single cells.

Various embodiments discussed herein comprise microfluidic devices thatare configured to encapsulate single particle or cells with highthrough-put. Various embodiments of the microfluidic devices can beconfigured to provide encapsulation efficiency of 30% or higher. Forexample, various microfluidic devices discussed herein can be configuredto encapsulate particles or cells having a size of about 2.5 μm withencapsulation efficiency of about 30% or greater in less than 1 second.Various innovative aspects of the subject matter of this applicationrely on various principles governing size based trapping in dropletgeneration region. Various embodiments of microfluidic devices discussedherein employ a simple trapping-encapsulating method to enhance theencapsulation efficiency with a low concentration of particles in thedispersed phase. Various embodiments of microfluidic devices discussedin this application can be configured as sorting devices that arecapable of sorting droplets with particles and without particles.

One innovative aspect of the subject matter described in this disclosurecan be implemented in a microfluidic device, comprising a first fluidchannel configured to transport a first fluid; a second fluid channelconfigured to transport a second fluid; an output fluid channel disposeddownstream from the first and the second fluid channels; and aflow-focusing region, the first and the second fluid channelsterminating in the flow-focusing region. The flow-focusing regioncomprises an orifice disposed to output the first and the second fluidstransported through the first and the second fluid channels into theoutput fluid channel. The flow-focusing region also can include a fluidcontroller that is configured to control the flow rate and/or disruptvortices. For example, the fluid controlled can be configured to adjustthe fluid pressure of the first and/or the second fluid such thatparticles or cells dispersed in the first or the second fluid can betrapped in a first mode and encapsulated in droplets of the first or thesecond fluid in a second mode. In various embodiments, the fluidcontroller can be configured to vibrate an air cavity placed upstreamfrom the orifice. The air cavity can be vibrated to modulate the flowrate. In other embodiments the flow rate can be modulated in other ways,such as by restricting or widening the passage upstream of the orificeor by modulating the flow generating device, e.g., a pump device. Themicrofluidic device can further comprise a piezo-electric transducerconfigured to vibrate the air cavity.

One innovative aspect of the subject matter described in this disclosurecan be implemented in a method of encapsulating a solid sample in adroplet. The method comprises flowing a first fluid through a firstfluid channel, the first fluid including the solid sample (e.g.,particle/cell/bead); flowing a second fluid through a second fluidchannel; forcing the first and the second fluids through an orifice intoan output fluid channel; and modulating a fluid flow parameter in a zoneupstream from the orifice. A flow rate of the second fluid is configuredto generate droplets of the first fluid of a desired size, and the fluidflow parameter is configured to achieve a desired encapsulationefficiency. The fluid flow parameter can be a vibration parameter andthe modulating can involve vibrating an air cavity. The fluid flowparameter can be a flow rate parameter and the modulating can involvealtering the fluid flow rate upstream from the orifice. In variousembodiments, a piezo-electric transducer can be configured to vibratethe air cavity. In various embodiments, the solid sample can comprise acell or a bead including an organic material. The parameter can be afrequency of vibration or an amplitude of vibration.

Another innovative aspect of the subject-matter described in thisdisclosure can be implemented in a microfluidic device including one ormore outer channels configured to transport a first fluid towards anoutput channel and one or more inner channels configured to transport asecond fluid towards the output channel. The microfluidic devicecomprises a fluid control system to control the flow rate of the firstfluid through the outer channels in order to first trapparticles/cells/beads that are larger than a certain size (or diameter)immersed in the second fluid. If the flow rate of the first fluid isabove a certain flow rate, two symmetric vortices (or microstreaming)form right before the orifice in the second fluid streaming through theinner channels. The inner channels contain the particles/cells/beads.These vortices can trap particles/beads/cells above a certain size andallow smaller ones to leak through a gap between the vortices and thechannel walls having a dimension d_(gap). If a particle/bead/cell has aradius larger than the d_(gap), the probability that theparticle/bead/cell is trapped in the vortices is high. Accordingly, thefluid control system can be configured to control the flow rate of thefirst fluid such that vortices are formed, then theparticles/beads/cells in the second fluid can be trapped. The fluidcontrol system can be configured to lower the flow rate of the firstfluid such that the size of the vortices is reduced or the vortices maynot be even formed so as to allow incoming particles/beads/cells to flowthrough the orifice into the output channel. By controlling the flowrate of the first fluid, the incoming particles/beads/cells can beencapsulated in droplets of the second fluid that are formed when thesecond fluid is forced through the orifice in the output channel. Inthis manner, the flow control system can be used to switch themicrofluidic device between a trapping mode and an encapsulation mode.By accumulating solid sample (e.g., particles/beads/cells) in thetrapping mode first and then releasing the solid sample (e.g.,particles/beads/cells) in the encapsulation mode, higher percentage ofencapsulation a single particle/cell/bead in a single droplet is madepossible.

One innovative aspect of the subject matter of this application isembodied in a microfluidic device, comprising: a first fluid channelconfigured to transport a continuous phase; a second fluid channelconfigured to transport a dispersed phase, the dispersed phasecomprising a solid sample having a plurality of particles; a dropletgeneration region; and a fluid controller. The droplet generation regioncomprises a mixing region configured to receive the continuous phase andthe dispersed phase; and an output fluid channel connected to the mixingregion through an orifice. The fluid controller is configured to adjustat least one flow parameter of the continuous phase or the dispersedphase to trap the plurality of particles of the dispersed phase in themixing region in a first mode such that the plurality of particles ofthe dispersed phase are prevented from flowing through the orifice. Thefluid controller is further configured to adjust at least one flowparameter of the continuous phase or the dispersed phase to allow theplurality of particles to flow through the orifice such that theplurality of particles are encapsulated in droplets of dispersed phasein a second mode.

In various embodiments of the microfluidic device, the at least one flowparameter can include a flow velocity and/or a fluid pressure. In thefirst mode, the fluid controller can be configured to adjust at leastone flow parameter of the continuous phase or the dispersed phase togenerate a vortex in a flow field of the dispersed phase in the mixingregion. In the first mode, the fluid controller can be configured toadjust at least one flow parameter of the continuous phase or thedispersed phase such that a distance (d_(gap)) between an outermoststreamline of the vortex generated in flow field of the dispersed phaseand an interface between the dispersed phase and the continuous phase isgreater than or equal to a size of the plurality of particles. In thesecond mode, the fluid controller can be configured to adjust at leastone flow parameter of the continuous phase or the dispersed phase todissipate vortices in a flow field of the dispersed phase in the mixingregion. In various embodiments, the continuous phase can comprise alipid and/or the dispersed phase can comprises an aqueous material. Invarious embodiments, the plurality of particles can comprise biologicalcells or molecules. The size of the plurality of particles can be about2.5 μm.

Various embodiments of the microfluidic device can further comprise athird fluid channel configured to allow the flow of a buffer solutionthrough the mixing region. A parameter of the flow of the buffersolution can be controlled by the fluid controller to untrap particleshaving a size smaller than a desired size from the plurality ofparticles.

One innovative aspect of the subject matter of this application includesa method of encapsulating a solid sample in a droplet. The methodcomprises: flowing a continuous phase through a first fluid channel at afirst flow rate; flowing a dispersed phase through a second fluidchannel at a second flow rate, the dispersed phase comprising particlesor cells; trapping the particles or cells in a mixing region thatreceives the dispersed phase and the continuous phase; and reducing thefirst flow rate to encapsulate the trapped particles or cells indroplets of the dispersed phase generated when the dispersed phase andthe continuous phase exit the mixing region through an orifice. A sizeof the particles or cells can be less than or equal to a distance (dgap)between an outermost streamline of a vortex formed in flow field of thedispersed phase and an interface between the dispersed phase and thecontinuous phase.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects and advantages are described belowwith reference to the drawings, which are intended to illustrate but notto limit the inventions. In the drawings, like reference charactersdenote corresponding features consistently throughout similarembodiments. The following is a brief description of the drawings.

FIGS. 1A and 1B illustrate embodiments of a microfluidic deviceincluding lateral cavity acoustic transducers (LCATs) that areconfigured to manipulate fluids.

FIG. 2 schematically illustrates an embodiment of a microfluidic deviceincluding a flow-focusing droplet generator.

FIG. 3 illustrates the vortices generated in the flow-focusing regionaround the orifice of an embodiment of a microfluidic device.

FIG. 4 schematically illustrates a design of an embodiment of amicrofluidic droplet generating device including an air cavityconfigured to disrupt vortices.

FIG. 5A illustrates an embodiment of a microfluidic device that isconfigured to encapsulate particles or cells in droplets of a dispersedphase that are suspended in a continuous phase. FIG. 5B illustratesdetails of the droplet generation region of the microfluidic devicedepicted in FIG. 5A.

FIG. 6A is a simulation showing the generation of vortices in thedroplet generation region of an embodiment of a microfluidic device.FIG. 6B is a simulation showing streamline fluid flow in the dropletgeneration region of an embodiment of a microfluidic device.

FIG. 7 illustrates an embodiment of a microfluidic device in which theparticles or cells are trapped in the droplet generation region.

FIG. 8A shows formation of droplets in the geometry-controlled regime.

FIG. 8B shows the formation of droplets in the dripping regime. FIG. 8Cshows the formation of droplets in the jetting regime.

FIG. 9A shows particles or cells trapped in the droplet formation regionof a microfluidic device. FIG. 9B shows the encapsulation of theparticles or cells in single droplets. FIG. 9C shows the particles orcells approximately 1 second after encapsulation as depicted in FIG. 9B.

FIG. 10 illustrates a simulation of fluid flow through an embodiment ofa microfluidic device.

FIG. 11 is a plot of the droplet encapsulation efficiency as a functionof a parameter that depends on droplet size and frequency.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

This application is directed to improve cell/bead encapsulationefficiency using flow parameter modulation such as with a fluidcontroller that is configured to vary flow velocity and/or fluidpressure or an acoustically resonant structure that reduces cell/beadtrapping in a flow-focusing region of a droplet generation region of amicrofluidic device.

Lateral Cavity Acoustic Transducer (LCAT)

The lateral cavity acoustic transducer (LCAT) is a microfluidic actuatorconfigured to carry out diverse functions such as microfluidic pumping,mixing, and particle trapping.

The LCAT is based on a phenomenon that uses trapped air bubbles inliquid for various applications including but not limited to pumping,mixing, particle trapping, particle sorting, bead deflection, etc. Theair bubbles are trapped in sidewall lateral cavities and are excited byan external acoustic field which causes the liquid/air interface toresonate. As the air/liquid interface resonates, a net force is producedout of the end of the cavity. By controlling the angle of thesecavities, different fluidic operations can be performed. For example, byorienting the LCATs at an oblique angle to the microfluidic channel,fluid pumping or propulsion can be accomplished. As another example, byorienting the LCATs perpendicular to the microfluidic channel, fluidmixing can be accomplished. FIGS. 1A and 1B illustrate embodiments of amicrofluidic device including LCATs that are configured to manipulatefluids. Without subscribing to any theory, exciting the trapped airbubbles in the LCATs by an acoustic field can induce microstreaming inthe microfluidic channel that can be used for various applicationsincluding but not limited to pumping, mixing, particle trapping,particle sorting, bead deflection, etc.

In various implementations, the air bubbles trapped in the sidewalllateral cavities can be excited using piezo-electric transducers. Insuch implementations, the vibrating air cavity/bubble can inducemicrostreaming of the liquid flowing through the microfluidic channel.

Microfluidic Droplet Generators

Microfluidic devices including droplet generation portions can be usedto create droplets of a fluid (e.g., oil or water). Microfluidic devicesthat include droplet generation portions can be used: to study chemicalreactions, in drug delivery, in drug discovery, etc. One method ofgenerating droplets in microfluidic devices uses flow focusing. FIG. 2schematically illustrates an embodiment of a microfluidic deviceincluding a flow-focusing droplet generator. The flow-focusing dropletgenerator generates droplets by flowing a first liquid (e.g., water)through the channel 205 and a second liquid (e.g., oil) through thechannels 207 a and 207 b. The first and the second liquid streams areforced through an orifice 209. The first liquid flowing through thechannel 205 (e.g., water) is broken up to form discrete droplets as aresult of shear forces. The size of the generated first liquid dropletsgenerated can depend on a variety of factors including velocity of thesecond liquid. For example, as the velocity of the second liquid isincreased, the size of the first liquid droplets is reduced. As the rateof droplet generation is increased and/or the size of the droplets isdecreased, vortices (e.g., microvortices) can be generated in theflow-focusing region around the orifice 209. FIG. 3 illustrates thevortices 305 a and 305 b generated in the flow-focusing region aroundthe orifice 209 of an embodiment of a microfluidic device.

The flow-focusing droplet generator can also be used to compartmentalizeor encapsulate a single cell or a bead comprising single cell, cellularmaterial or some other biological material in a single water droplet.Droplets encapsulating a single cell or bead can be useful for singlecell assays of cells (e.g., cancer cells or immune cells) that exhibitbiological heterogeneity for which assays that provide a populationaverage may be insufficient. As the rate of droplet generation isincreased and/or the size of the droplets is decreased, incoming cellsand/or beads may get trapped in the vortices that are generated in theflow-focusing region around the orifice 209 and not be encapsulated indroplets. This may result in a decrease in the percentage of cells orbeads that are encapsulated in the droplets. This disclosurecontemplates positioning a vibrating air cavity in the flow-focusingregion upstream from the orifice 209 to disrupt the particle-trappingvortices and facilitate cell/bead encapsulation. The vibrating aircavity can produce vortices in the flow-focusing region around theorifice 209 that are in a direction opposite to the direction of thevortices produced in the flow-focusing region around the orifice 209 asa result of increase in rate of droplet generation and/or decrease inthe size of the generated droplets.

FIG. 4 illustrates a device design created in L-Edit and AdobeIllustrator, and sent to CadArt Services to be printed as a 20 k DPItransparency mask. The mask was used to create an SU-8 mold on a siliconwafer. The mold, in turn, was used to cast devices made of PDMSelastomer. Liquids are flowed into the device through Tygon tubing, withflows controlled by either Harvard syringe pumps or pressure regulators.Air is caught in the structure 405 upstream from the orifice 209 as thedevice is filled with liquid. During droplet generation, the air trappedin the cavity 405 is vibrated by placing the device on a piezo-electrictransducer driven with a function generator and amplifier. As discussedabove, the vibrations can produce vortices that flow counter to thevortices in front of the orifice, allowing incoming cells/beads to enterthe produced droplets. Vibration parameters such as amplitude and/orfrequency can be adjusted to disrupt vortices that may be generated inthe flow-focusing region. The device design illustrated in FIG. 4 andthe device discussed above can reduce cell/bead trapping upstream fromthe orifice and improve/increase encapsulation efficiency. The devicedesign illustrated in FIG. 4 and the device discussed above can alsofacilitate switching between trapping and encapsulating modes bycontrolling piezoelectric transducer activation. As discussed below, thefunctionality of switching between trapping and encapsulating modes canbe advantageous in reducing the number of droplets that do not includeany cells or include multiple cells.

An objective of droplet microfluidic systems and devices is to directmolecules, particles or cells at a one-to-one ratio as droplets aregenerated in microchannels. The process of loading particles or cellsinto drops can be random and dictated by Poisson statistics. Theprobability of a drop containing k cells is (λ^(k) e^(−λ))/k!, where λis the average number of particles or cells per drop. Thus, the ratio ofdrops containing one particle or cell to those containing two particlesor cells is 2/λ. In order to reduce the number of drops that containmore than a single particle or cell the average loading densities shouldbe reduced. This can increase the probability that many drops mayencapsulate no particles or cells. Thus, in accordance with Poisson'sstochastic distribution, the resultant encapsulations are either oneswith multiple particles per droplet or ones with many empty droplets.Recent research indicates that there is a size separation similar toLCAT vortices. Thus integrating air cavities (e.g., LCATs) with dropletmicrofluidic systems can be advantageous in overcoming the limitation ofPoisson distribution (large number of empty droplets). For example, thecell/bead encapsulation efficiency can be increased by switching flowrate regimes away from flow rates at which cells/beads aretrapped/accumulated to flow rates at which cells/beads arereleased/encapsulated. The switching of the flow rates can beaccomplished by controlling the piezo-electric transducer that excitesthe air cavity/bubbles in the LCATs.

FIG. 5A illustrates an embodiment of a microfluidic device 500 that canencapsulate particles or cells in droplets of a dispersed phase that aresuspended in a continuous phase. The continuous phase and the dispersedphase comprise immiscible materials. For example, in some embodimentsthe continuous phase can comprise a lipid and the dispersed phase cancomprise an aqueous material. As another example, in some embodimentsthe continuous phase can comprise an aqueous material and the dispersedphase can comprise a lipid.

In the illustrated embodiment, the microfluidic device 500 includes afirst inlet 502 for introducing a first material that provides thecontinuous phase, a second inlet 506 for introducing a second materialthat is immiscible in the first material and provides the dispersedphase, a droplet generating region 514 and an outlet 518. The secondmaterial also includes a solid sample (e.g., particles or cells orbeads) that are to be encapsulated. In various embodiments, a lipidphase (e.g., an oil, a fatty acid, etc.) can be introduced through thefirst inlet 502 and an aqueous phase (e.g., water) including the solidsample (e.g., particles or cells or beads) can be introduced through thesecond inlet 506. In various embodiments, the dispersed phase cancomprise blood and the continuous phase can comprise materials that haveappropriate viscosity and provide equilibrium surface tension betweenthe continuous and dispersed phases such that droplets are formed in thedripping regime as discussed below. The first material (e.g., a lipid oran aqueous material) introduced through the first inlet 502 istransmitted towards the droplet generating region 514 through themicrofluidic channels 504 a and 504 b and the second material (e.g., anaqueous material or a lipid) including the solid sample (e.g., particlesor cells or beads) introduced through the second inlet 506 istransmitted towards the droplet generating region 514 throughmicrofluidic channels 508 a, 508 b and 512. FIG. 5B illustrates thedetails of the droplet generating region 514. The droplet generatingregion 514 includes a mixing region 513 that is fluidically connected toan enlarged region 522 through an orifice 520, and an outletmicrofluidic channel 516 connected to the outlet 518. The first and thesecond materials (e.g., aqueous material and the lipid) enter theenlarged region 522 through the orifice 520 and droplets of the secondmaterial (e.g., aqueous material) are formed in the first material(e.g., lipid) as the first and the second materials exit through theorifice 520.

As depicted in FIG. 5B, the droplet generating region 514 furtherincludes a post 524 that is configured to change the flow field (e.g.,change the direction of flow of the first and the second material,change the flow velocity of the first and the second material or both)and/or decrease the vortex sizes in the droplet generation region 514.

Referring back to FIG. 5A, the microfluidic device 500 further comprisesa buffer region that includes a third inlet 510 for introducing a buffersolution (e.g., water) and microfluidic channel 511. The solid sample(e.g., particles or cells or beads) introduced through the second inlet506 can be encapsulated in droplets of the second material that areformed as the first and the second materials exit through the orifice520.

Flow parameters (e.g., flow velocity and/or fluid pressure) of thecontinuous and/or the dispersed phase can be adjusted to trap the solidsample (e.g., particles, cells or beads) introduced through the secondinlet 506 are trapped in the mixing region 513. A buffer solution (e.g.,water) can be made to flow through the mixing region 513. The flowparameters (e.g., flow velocity and/or fluid pressure) of the buffersolution can be adjusted to wash away unwanted portions of the solidsample (e.g., particles, cells or beads having a size less than adesired size) and/or debris from the trapping vortices that areconfigured to trap the solid sample (e.g., particles or cells or beads).After the vortices have been washed, the flow parameters (e.g., flowvelocity and/or fluid pressure) of the continuous and/or dispersed phasecan be adjusted to release the desired portion of the solid sample(e.g., particles or cells or beads of a desired size) such that thedesired portion of the solid sample can be encapsulated in droplets ofthe second material. In various embodiments, the trapping of the desiredportions of the solids sample and washing of the vortices by the buffersolution can advantageously increase the concentration of the desiredportion of the solid sample. Various embodiments of the microfluidicdevice can be configured to intermittently switch between flowing thedispersed phase and flowing the buffer solution. Switching betweenflowing the dispersed phase and the buffer solution can be advantageousin flushing the vortices to remove particles having undesirable size anddebris and to increase concentration of particles having desirable sizeas discussed above. Another advantage of switching between flowing thedispersed phase and the buffer solution can be to prevent oversaturationof the vortices.

The microfluidic device can include a fluid controller that isconfigured to control various fluid parameters of the buffer solution,the first material, the second material and/or the particle or cells.For example, the fluid controller may be configured to control the flowrates of the first material, the second material and/or the particle orcells. As another example, the fluid controller may be configured tocontrol the fluid pressure of the first material, the second materialand/or the particle or cells.

The fluid flow of the first material and the second material through themicrofluidic channels of the microfluidic device 500 can be simulatedusing a computer program (e.g., Fluent). The fluid flow of the first andthe second materials can be simulated by considering the flow as a 2Dgeometry. Depending on the flow velocity of the first and the secondmaterials two kinds of flow fields are generated—a flow field withvortex and a flow field without vortex.

When the pressure of external phase (e.g., continuous phase or firstmaterial) is relatively high, the velocity of external phase (e.g.,continuous phase or first material) can also be large. This in turn canmake the velocity of the internal flow (e.g., the dispersed phase or thesecond material with or without the particles or cells) near theinterface large as well. When the flow rate of the internal flow (e.g.,the dispersed phase or the second material with or without the particlesor cells) near the interface is equal to the flow rate of internal phasedroplets (e.g., droplets of the dispersed phase or droplets of thesecond material with or without the particles or cells) at or near theorifice 520, vortices can be formed in the flow field of the internalphase (e.g., the dispersed phase or the second material with or withoutthe particles or cells) in the droplet generation region 514 (e.g., inthe mixing region 513). The formation of the vortices may reduce theflow rate of the internal phase (e.g., the dispersed phase or the secondmaterial with or without the particles or cells) at the orifice 520.

When the pressure of external phase (e.g., continuous phase or firstmaterial) is relatively low, the flow rate of internal flow (e.g., thedispersed phase or the second material with or without the particles orcells) near the interface is less than the flow rate of internal-phasedroplets (e.g., droplets of the dispersed phase or droplets of thesecond material with or without the particles or cells) at or near theorifice 520 such that no vortices are generated at or near the orifice520 and the flow rate of the internal flow (e.g., the dispersed phase orthe second material with or without the particles or cells) towards theorifice 520 may not be reduced.

FIG. 6A shows the generation of vortices 605 in the fluid flow of thedispersed phase or the second material with or without the particles orcells in the droplet generation region 514 when the pressure of thecontinuous phase or the first material is high and FIG. 6B shows astreamlined fluid flow (e.g., without vortices) of the dispersed phaseor the second material with or without the particles or cells in thedroplet generation region 514 when the pressure of the continuous phaseor the first material is low.

The particles or cells introduced through the third inlet 510 will notbe trapped in the mixing region 513 in the absence of any vortices influid flow of the dispersed phase or the second material at or near theorifice 520. However, when vortices are formed in the fluid flow of thedispersed phase or the second material at or near the orifice 520, beadsmay be trapped depending on the distance between the interface of thecontinuous and the dispersed phase and the outermost streamline of thevortex. The distance between the interface of the continuous and thedispersed phase—indicated by reference numeral 615 in FIG. 6A and theoutermost stream-line 610 of the vortex 605 is referred to herein asd_(gap). The distance between the interface of the continuous and thedispersed phase and the outermost stream-line of the vortex d_(gap) candepend on the flow velocities of the continuous and the dispersedphases. When the radius of particles or cells is greater than d_(gap),the center of the particles or cells will cross into a closed streamline(e.g., a streamline of the vortex) increasing the probability that theparticles or cells are trapped in the mixing region 513. However, whenthe radius of particles or cells is less than d_(gap), some particles orcells may be trapped for some time in the mixing region 513 due toinertia of the particles or cells or some other forces. But theseparticles or cells may eventually go through the orifice 520 sometimeafter they have been trapped.

FIG. 7 illustrates an embodiment of a microfluidic device in which theparticles or cells are trapped in the mixing region 513 of the dropletgeneration region 514 when d_(gap) is less than the size (e.g., radius)of particles or cells. To increase the encapsulation efficiency of theparticles or cells, it may be advantageous if the distance between theinterface of the continuous and the dispersed phase and the outermoststream-line of the vortex d_(gap) is greater than or equal to the size(e.g., radius) of the particles or cells.

There are mainly three kinds of droplet formation regimes:geometry-controlled region, dripping regime and jetting regime. Thedroplet formation regime is determined by the capillary numberCa=μV/γ_(EQ), where μ is the viscosity of the continuous phase, V is thesuperficial velocity of the continuous phase, and γ_(EQ) is theequilibrium surface tension between the two continuous and the dispersedphases.

Most traditional flow-focusing devices have been operated in thegeometry-controlled regime, termed for the large dependence of dropletsize on the smallest feature size in the device (e.g., the orifice). Inthis regime droplets break off from the dispersed phase finger followinga protrude-and-retract mechanism. Droplets in the geometry-controlledregime can be highly monodisperse but limited in minimum size by thewidth of the orifice.

An increase in the capillary number Ca can lead to droplet generation inthe dripping regime. This regime produces monodisperse droplets smallerthan the size of the orifice due to narrowing of the dispersed phasefinger. The dripping mode can be characterized by a dispersed phase tipthat does not retract but rather remains at a fixed location in theorifice, generating a stream of droplets off the tip due to Rayleighcapillary instability.

A further increase in the capillary number leads to droplet generationin the jetting mode, wherein the dispersed phase finger extends far intothe post-orifice channel (e.g., the enlarged region 522 and/or thechannel 516). Droplets, which break off the tip of the dispersed phasefinger due again to Rayleigh capillary instability, tend to be as largeas or larger than the orifice width in the jetting mode and may bepolydisperse.

FIGS. 8A, 8B and 8C show representative images of three distinct dropletformation regimes. FIG. 8A shows droplet formation in thegeometry-controlled regime. FIG. 8B shows droplet formation in thedripping regime and FIG. 8C shows droplet formation in the jettingregime.

To encapsulate single particles or cells into one droplet, it may beadvantageous if the size (e.g., the radius) of the particles or cells isalmost equal to d_(gap) without exceeding the size of d_(gap) such thatencapsulation of two or more particles into one droplet can be avoided.As discussed elsewhere herein, d_(gap) sets the threshold for trappingof cells at the cell sizes of interest. With this d_(gap), thecells/particles smaller than a certain size will be released in additionto concentrating the targeted cell size while the solution continues toflow through. If the radius of a bead or other solid sample is muchsmaller than d_(gap), it is more likely for clusters of multiple beads(or other solid sample) to get caught up in one droplet as it ispinched-off (separated). On the other hand if the bead (or solid sample)radius is slightly below d_(gap), only one bead (or solid sample) shouldbe encapsulated at a time. It may be further advantageous if the valueof d_(gap) remained constant.

In the geometry-controlled regime, because of the protrude-and-retractmechanism, the interface of two continuous and the dispersed phases willchange and vibrate during droplet formation process. This can also causethe size of the vortices and the size of d_(gap) to change, which canmakes it difficult to control the encapsulation.

In the dripping regime, the dispersed phase tip remains at a fixedlocation in the orifice, generating a stream of droplets off the tip dueto Rayleigh capillary instability. The interface of the continuous andthe dispersed phase can have a steady shape in the dripping regime. Thesize of the vortices and d_(gap) can also be constant in the drippingregime. Also, the dripping regime produces very small monodispersedroplets with high throughput, which can allow d_(gap) to have a valueapproximately equal to the size (e.g., radius) of the particles or thecells.

The interface between the continuous phase and the dispersed phase canbe steady in the jetting regime. However, the size of droplets may notbe constant and may be polydisperse. Thus, the dripping regime can haveappropriate d_(gap) for encapsulation of single particles or cells in asingle droplet and also produce monodisperse droplet may be the mostsuitable droplet formation process for encapsulating single particles orcells in a single droplet.

To test the encapsulation performance of the microfluidic device 500 inthe dripping droplet formation regime, ethyl oleate and 2% ABIL EM 90was used as the continuous phase, and mixture of water, lipid, glyceroland surfactant was used as the dispersed phase. For example, thedispersed phase can include 5 mg DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine, Avanti Polar Lipids) and1.96 mgDSPE-PEG2000(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethyleneglycol)-2000], Avanti Polar Lipids) that are combined in a glass vialand dissolved in chloroform (CHCl3, Sigma) to form a homogeneousmixture. The solvent can be evaporated with a nitrogen stream. 4 mL ofultra-pure water can be added to the dry lipid mixture and sonicated at50° C. for 20 minutes. The solution was combined with an additional 4 mLof glycerol (Sigma), and 2 mL of nonionic surfactant (Pluronic F-68,Sigma), sonicated at 50.0 for 20 minutes. The lipid solution wassonicated again for 15 minutes immediately prior to use to reduceunwanted liposome formation. The continuous phase is introduced throughthe first inlet 502 and the dispersed phase is added through the secondinlet 510. Particles (e.g., PeakFlow™ Green flow cytometry referencebeads, 2.5 m, Molecular Probes) having a size (e.g., radius) of 2.5 μmare introduced through the third inlet 506 into the dispersed phase.

Since the high-throughput of droplet formation, normal concentration ofparticles in dispersed phase may cause very low encapsulationefficiency, a method to get high concentration of particles or cells indispersed phase as described below can be used to perform encapsulationof single particles in a short time interval.

To get high concentration of particles or cells in the dispersed phase,the flow parameters (e.g., flow velocity and/or pressure of thecontinuous and the dispersed phases) can be adjusted using the fluidcontroller to generate vortices in the dispersed phase such that d_(gap)is less than the size (e.g., radius) of the particles or cells so thatall the particles or cells can be trapped in the mixing region 513 ofthe droplet generation region 514, as shown in FIG. 9A. Then thepressure of continuous phase can be reduced using the fluid controllersuch that d_(gap) is greater than the size (e.g., radius) of theparticles or cells. The particles or cells that are trapped in thedroplet generation region 514 will be encapsulated in droplets in ashort time interval (e.g., in less than 1 second).

FIGS. 5B and 9A shows the particles or cells trapped in the mixingregion 513 of the droplet generation region 514 when d_(gap) is lessthan the size (e.g., radius) of the particles or cells. FIGS. 5B and 9Billustrates the encapsulation process results when d_(gap) is greaterthan the size (e.g., radius) of the particles or cells. FIGS. 5B and 9Cillustrates the encapsulation of the particles or cells in dropletsapproximately 1 second after d_(gap) is made greater than the size(e.g., radius) of the particles or cells.

With further reference to FIGS. 9A-9C, the width of the orifice (e.g.,orifice 520) is approximately 4 μm. Without subscribing to anyparticular theory, vortices can be disrupted by adjusting the flow rateand/or fluid pressure of the continuous phase and/or the dispersed phaseas discussed above. For example, as the flow rate and/or the fluidpressure of the continuous phase increases solid sample (e.g.,particles/cells/beads) having a size (e.g., radius) above a thresholdlength are trapped and do not flow through the orifice 520. If the flowrate and/or the fluid pressure of the continuous phase is lowered belowa certain threshold, the size of the vortices could reduce and/or thevortices may disappear. Under these flow conditions, the solid sample(e.g., particles/cells/beads) that were previously trapped and notallowed to flow through the orifice may flow through the orifice 520.Accordingly, in various embodiments, the flow rate and/or the fluidpressure of the continuous phase can be adjusted to reduce vortices inthe flow-focusing region as shown in FIG. 10. FIG. 10 illustrates asimulation of the fluid flow in the mixing region 513 of the dropletgeneration region 514 (depicted in FIG. 5B) of an embodiment of amicrofluidic device 500 (depicted in FIG. 5A) when the flow rate and/orthe fluid pressure of the continuous phase is reduced to reduce a sizeof the vortices and/or to dissipate vortices. It was observed thatvarying the flow rate and/or the fluid pressure of the continuous phasecan result in switching between trapping of the incoming solid sample(e.g., particles/cells/beads) and releasing of the incoming solid sample(e.g., particles/cells/beads) for encapsulation in droplets of thedispersed phase. As the flow rate and/or the fluid pressure of thecontinuous phase was adjusted to reduce vortices in the flow-focusingregion trapping of the incoming solid sample (e.g.,particles/cells/beads) was reduced which resulted in an increase indroplet encapsulation efficiency.

Encapsulation of particles or cells can be performed by varying initialconcentration (IC) (number of particles/nL) of particles or cell in thedispersed phase, real Concentration(RC) (number of particles/nL) oftrapped particles or cells in the droplet generation region beforeencapsulation, the initial droplet diameter(IDD) (μm) which correspondsto the diameter of droplets before encapsulation, final dropletdiameter(FDD) (μm) which corresponds to the diameter of droplets afterencapsulation, droplet formation frequency(DFF) (number of droplets/s)which corresponds to the number of droplets formed per second duringencapsulation, encapsulation efficiency(EE) which corresponds to theproportion of droplets with particles among all droplets formed

The encapsulation efficiency for different initial concentration of6.79, 15.65, 24, 27.69, 40 number of particles/nL and for an initialdroplet diameter (IDD) of about 4 μm is obtained. The initial dropletdiameter(IDD) of about 4 μm can provide a d_(gap) that is smaller thanthe size (e.g., radius) of the particles or cells and keep particles orcells from encapsulation. It is observed that the initialconcentration(IC) has nearly no effect on encapsulation efficiency (EE).Instead, for certain continuous and dispersed phases and in the drippingdroplet formation regime, the real concentration (RC), final dropletdiameter(FDD) and droplet formation frequency(DFF) determine theencapsulation efficiency(EE).

FIG. 11 below shows the relationship between real concentration (RC),final droplet diameter(FDD), droplet formation frequency(DFF) andencapsulation efficiency(EE). The coefficient of determination of

$\frac{{RC} \times {FDD}^{3}}{{DFF}^{1/2}}$

and EE is 0.63009, which indicates that the two parameters have amoderate positive correlation.

FIG. 11 is a plot of the droplet encapsulation efficiency as a functionof a parameter that is given by the equation

$\frac{({RealConcentration}) \times ({FinalDropletDiameter})^{3}}{({DropletFormationFrequency})^{1/2}}{( {\times 10^{- 2}s^{1/2}} ).}$

It is observed from FIG. 11 that the droplet encapsulation efficiencyincreases as the droplet diameter increases and/or the droplet formationfrequency decreases. It is noted that the size of the cells trapped candepend on the geometry (e.g. diameter) of the droplets generated.

As noted above, there is a positive correlation between realconcentration (RC) and encapsulation efficiency (EE). Also, finaldroplet diameter (FDD) shows the volume of the dispersed phase in onedroplet. So FDD³, which is a volume term, should be considered. Lastly,there is a negative correlation between droplet formation frequency(DFF) and encapsulation efficiency because of new coming dispersed phasewith initial concentration of particles. The power ½ is added to DFF inthe above equation to provide a higher correlation to encapsulationefficiency (EE).

Consequently, in order to enhance the encapsulation efficiency (EE), itmay be advantageous to increase real concentration (RC) and finaldroplet diameter (FDD) and decrease droplet formation frequency (DFF).

However, the real concentration (RC) may have a maximum limit. When RCis higher, trapped particles or cells may influence the flow field inthe droplet generation region such that droplet formation may becomeunstable. The regime may change from dripping to geometry-controlledregime, which may release trapped particles without trapping them.

When the final droplet diameter (FDD) is increased, d_(gap) may alsoincrease. It is more likely to encapsulate two or more particles intoone droplet under such conditions. When the droplet formation frequency(DFF) is decreased, the velocity at the interface V may also decreasewhich may reduce the capillary number Ca which can change the dropletformation region to be geometry-controlled.

Thus, in order to improve the encapsulation efficiency (EE), it may beadvantageous to maintain real concentration (RC) and final dropletdiameter (FDD) near their maximum limits and find materials for thecontinuous and dispersed phases that have appropriate viscosity of thecontinuous phase μ and equilibrium surface tension between thecontinuous and dispersed phases γ_(EQ) to perform dripping regime underrelatively low velocity V.

Various embodiments of the microfluidic device 500 can include twooutlets. A microswitch may be configured to direct dropletsencapsulating particles to the target outlet and direct other emptydroplets to the waste outlet. Such a device may also include a sortingsection that sorts droplets with and without particles prior to thesection including the microswitch.

A hydrodynamic method for high-throughput encapsulation of singleparticles with relatively high encapsulation efficiency in drop-basedmicrofluidic devices are discussed herein.

Although certain preferred embodiments and examples are discussedherein, it will be understood by those skilled in the art that theinnovative aspects extend beyond the specifically disclosed embodimentsto other alternative embodiments and/or uses of the innovative aspectsand obvious modifications and equivalents thereof. In addition, whileseveral variations of the innovative aspects have been shown anddescribed in detail, other modifications, which are within the scope ofthese inventions, will be readily apparent to those of skill in the artbased upon this disclosure. It is also contemplated that variouscombination or sub-combinations of the specific features and aspects ofthe embodiments may be made and still fall within the scope of theinventions. It should be understood that various features and aspects ofthe disclosed embodiments can be combined with or substituted for oneanother in order to form varying modes of the disclosed inventions.Further, the actions of the disclosed processes and methods may bemodified in any manner, including by reordering actions and/or insertingadditional actions and/or deleting actions. Thus, it is intended thatthe scope of at least some of the innovative aspects discussed hereinshould not be limited by the particular disclosed embodiments describedabove. The limitations in the claims are to be interpreted broadly basedon the language employed in the claims and not limited to the examplesdescribed in the present specification or during the prosecution of theapplication, which examples are to be construed as non-exclusive.

What is claimed is:
 1. A microfluidic device, comprising: a first fluidchannel configured to transport a first fluid; a second fluid channelconfigured to transport a second fluid; an output fluid channel disposeddownstream from the first and the second fluid channels; and aflow-focusing region, the first and the second fluid channelsterminating in the flow-focusing region, the flow-focusing regioncomprising: an orifice disposed to output the first and the secondfluids transported through the first and the second fluid channels intothe output fluid channel; and an air cavity placed upstream from theorifice.
 2. The microfluidic device, further comprising a piezo-electrictransducer configured to vibrate the air cavity.
 3. A method ofencapsulating a solid sample in a droplet, the method comprising:flowing a first fluid through a first fluid channel, the first fluidincluding the cell/bead; flowing a second fluid through a second fluidchannel; forcing the first and the second fluids through an orifice intoan output fluid channel; and modulating a fluid flow parameter in a zonedisposed upstream from the orifice, wherein a flow rate of the secondfluid is configured to generate droplets of the first fluid of a desiredsize, and wherein the fluid flow parameter is configured to achieve adesired encapsulation efficiency.
 4. The method of claim 3, wherein apiezo-electric transducer is configured to vibrate the air cavity. 5.The method of claim 3, wherein the solid sample comprises a cell.
 6. Themethod of claim 3, wherein the solid sample comprises a bead includingan organic material.
 7. The method of claim 3, wherein the parameter isa frequency of vibration.
 8. The method of claim 3, wherein theparameter is an amplitude of vibration.
 9. The method of claim 3,wherein modulating the fluid flow parameter includes vibrating an aircavity disposed upstream from the orifice, wherein a vibration parameterof the air cavity is configured to achieve a desired encapsulationefficiency.
 10. The method of claim 3, wherein modulating the fluid flowparameter includes altering the fluid flow rate upstream from theorifice.
 11. The method of claim 3, wherein modulating the fluid flowparameter includes applying a standing wave to the first fluid channeland/or to the second fluid channel, the standing wave having awavelength a multiple of (e.g., two, four, six, or more times) a widthof the first fluid channel and/or to the second fluid channel.
 12. Amicrofluidic device, comprising: a first fluid channel configured totransport a continuous phase; a second fluid channel configured totransport a dispersed phase, the dispersed phase comprising a solidsample having a plurality of particles; a droplet generation regioncomprising: a mixing region configured to receive the continuous phaseand the dispersed phase; and an output fluid channel connected to themixing region through an orifice; and a fluid controller configured to:adjust at least one flow parameter of the continuous phase or thedispersed phase to trap the plurality of particles of the dispersedphase in the mixing region in a first mode such that the plurality ofparticles of the dispersed phase are prevented from flowing through theorifice; and adjust at least one flow parameter of the continuous phaseor the dispersed phase to allow the plurality of particles to flowthrough the orifice such that the plurality of particles areencapsulated in droplets of dispersed phase in a second mode.
 13. Themicrofluidic device of claim 12, wherein the at least one flow parameterincludes a flow velocity.
 14. The microfluidic device of claim 12,wherein the at least one flow parameter includes a fluid pressure. 15.The microfluidic device of claim 12, wherein in the first mode, thefluid controller is configured to adjust at least one flow parameter ofthe continuous phase or the dispersed phase to generate a vortex in aflow field of the dispersed phase in the mixing region.
 16. Themicrofluidic device of claim 15, wherein in the first mode, the fluidcontroller is configured to adjust at least one flow parameter of thecontinuous phase or the dispersed phase such that a distance (d_(gap))between an outermost streamline of the vortex generated in flow field ofthe dispersed phase and an interface between the dispersed phase and thecontinuous phase is greater than or equal to a size of the plurality ofparticles.
 17. The microfluidic device of claim 12, wherein in thesecond mode, the fluid controller is configured to adjust at least oneflow parameter of the continuous phase or the dispersed phase todissipate vortices in a flow field of the dispersed phase in the mixingregion.
 18. The microfluidic device of claim 12, wherein the continuousphase comprises a lipid.
 19. The microfluidic device of claim 12,wherein the dispersed phase comprises an aqueous material.
 20. Themicrofluidic device of claim 12, wherein the size of the plurality ofparticles is about 2.5 μm.
 21. The microfluidic device of claim 12,further comprising a third fluid channel configured to allow the flow ofa buffer solution through the mixing region, a parameter of the flow ofthe buffer solution being configured to untrap particles having a sizesmaller than a desired size from the plurality of particles.
 22. Amethod of encapsulating a solid sample in a droplet, the methodcomprising: flowing a continuous phase through a first fluid channel ata first flow rate; flowing a dispersed phase through a second fluidchannel at a second flow rate, the dispersed phase comprising particlesor cells; trapping the particles or cells in a mixing region thatreceives the dispersed phase and the continuous phase; and reducing thefirst flow rate to encapsulate the trapped particles or cells indroplets of the dispersed phase generated when the dispersed phase andthe continuous phase exit the mixing region through an orifice.
 23. Themethod of claim 22, wherein a size of the particles or cells is lessthan or equal to a distance (d_(gap)) between an outermost streamline ofa vortex formed in flow field of the dispersed phase and an interfacebetween the dispersed phase and the continuous phase.